Human diabetes, a disease in which a major indicator is an elevated blood glucose level, is generally believed to result from low insulin levels and elevated glucagon levels. However, hyperglycemia in non-insulin dependent diabetes, in both non-obese and obese patients, has been shown in the presence of both elevated glucagon and insulin levels.
Insulin is known to rapidly decrease blood glucose levels while glucagon, a polypeptide hormone twenty-nine amino acid residues in length, is believed to contribute to elevated blood glucose levels by binding to liver membrane receptors, and thereby triggering glycogenolysis, which results in the production of glucose. Elevated glucagon levels are also associated with a substantial increase in gluconeogenesis.
While stable control of insulin levels is difficult to achieve, treatment for insulin-dependent diabetes and some non-insulin dependent diabetes has been achieved through a combination of controlled diet and periodic doses of exogenous insulin. It is believed that the therapeutic use of glucagon antagonists will inhibit glycogenolysis and help to lower blood glucose levels in diabetics. These antagonists have the ability to bind to the glucagon receptor in the liver membrane, but are incapable of stimulating adenylate cyclase activity. The binding of glucagon to its cellular receptor is believed to trigger the stimulation of adenylate cyclase activity resulting in the production of cyclic AMP (cAMP), and results in an increase in glycogenolysis and its accompanying release of glucose. The glucagon-stimulated increase in inositol triphosphate, which acts as a signal for the release of calcium.sup.2+ sequestered in the endoplasmic recticulum, has been reported by Wakelam et al. (Nature 323:68-71, 1986), Unson et al. (Peptides 10:1171-1177, 1989) and Pittner and Fain (Biochem. J. 277:371-378, 1991).
Current methods for developing glucagon antagonists have relied on the development of specific glucagon analogs through the deletion or substitution of specific amino acids using solid-phase peptide synthesis, and high-level purification of these glucagon analogs through solid-phase synthesis methods in combination with other chromatographic methods. See, for example, Unson et al. (Peptides 10:1171-1178, 1989), Andreu and Merrifield (Eur. J. Biochem. 164:585-590, 1987), Gysin et al. (Biochemistry 25:8278-8284, 1986), Merrifield (U.S. Pat. No. 4,879,273) and Hruby (U.S. Pat. No. 4,430,326). These methods, however, do not lend themselves to the high through-put screening of large numbers of glucagon analogs.
There exists a need in the art for a method of detecting glucagon antagonists that does not rely upon the high-purity, solid-phase synthesis of glucagon analogs. The present method, through the use of recombinant DNA methods, permits the production of high numbers of glucagon analogs for screening through high through-put antagonist screening assays.